ABSTRACT Past studies on extrinsic signals and intrinsic transcription factors (TFs) have greatly enhanced our understanding of how the fate specification and differentiation of diverse neuronal cell types in CNS are genetically coded. The recent identification of diverse chromatin regulatory marks and the enzymes producing those marks allows us to integrate the potentially vital action of chromatin regulation with the activity of TFs in CNS development. However, this effort is riddled with a few challenges. 1) The developing CNS consists of profoundly heterogeneous cells at different developmental states, making it difficult to study how chromatin changes are orchestrated in each cell type. 2) While cell type-specific regulatory elements (herein referred to as cis-elements) are predicted to undergo the most functionally critical chromatin changes throughout development, such cis-elements are globally ill-defined. 3) Although multiple chromatin regulatory factors would be mobilized to cell type-specific cis-elements throughout development of each cell type, it is tough to identify the specific chromatin factors in action. Building on our prior effort to solve these limitations, the current study of the gene regulatory networks for spinal motor neuron (MN) development is a pioneering study in addressing the critical issue of chromatin regulation in CNS development. Our findings in the past funding cycles identified transcription codes and gene regulatory elements that underlie commitment and specification of MN fate, establishing an ideal cellular model to investigate chromatin regulation in CNS development. In the developing spinal cord, Olig2, a basic helix-loop-helix (bHLH) TF expressed in progenitors for MNs (pMNs), plays essential roles in establishing the pMN domain and keeping pMN cells from prematurely differentiating to MNs. As pMN cells begin to differentiate to MNs, the LIM homeodomain (HD) TFs Isl1 and Lhx3 are upregulated, along with the bHLH TF Ngn2. Isl1 and Lhx3 form a complex (Isl1-Lhx3), which directs MN fate via synergistic transactivation of MN genes with Ngn2. In this proposal to integrate these genetic programs with chromatin regulation, we hypothesize that during MN development, coordinated actions of the cell type-specific TFs (Olig2, Ngn2 and Isl1-Lhx3) and the chromatin modifiers (Ezh2, Jmjd3 and CBP/p300) orchestrate the chromatin changes in MN genes from transcriptionally poised/repressive to active state, enabling the timely acquisition of MN fate and cell differentiation. Based on our preliminary data, we specifically postulate that, in pMN cells, Olig2 recruits Ezh2 to MN genes, instituting the transcriptionally repressive chromatin mark, trimethylated histone H3-lysine 27 (H3K27me3). As Olig2 expression declines in differentiating pMN cells, Isl1-Lhx3 and Ngn2 seize MN-specific enhancers and recruit Jmjd3, which removes H3K27me3 and allows CBP/p300 to set up acetylated H3K27 (H3K27ac), a transcriptionally active chromatin mark. We will test our hypothesis using an ensemble of cellular, biochemical, genetic and genome-wide approaches.